Retroviral integrase (IN) catalyzes the essential step of integrating the double-stranded DNA copy of the viral genome into the chromosome of an infected cell. However, certain mutations of HIV-1 IN can impair reverse transcription, uncoating, and core morphology, and the underlying mechanisms for these defects are not well understood. We have characterized HIV-1 IN mutations that resulted in non-infectious viruses with low core yield and poor stability, and impairment at the step of early reverse transcription. Further analyses indicate that IN enhances core incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to capsid (CA) and promotes optimal stability of the viral core. We hypothesize that HIV-1 IN can impact uncoating by interacting with CA, which in turn affects the incorporation of CypA to the core. We have preliminary data indicating that IN interacts with CA within HIV-1 cores. We have further found that purified IN does not bind monomeric or dimeric CA, but can readily bind in vitro assembled core-like structures formed with purified CA or CA-NC, as well as a CA mutant that preferentially forms hexamers under non-reducing conditions. The goal of this application is to gain a better understanding of the interaction between HIV-1 IN and CA and the functional role of the IN-CA interaction during HIV-1 replication. The specific aims are (1) to characterize the physical interaction between HIV-1 IN and higher-order CA structures, and (2) to visualize the morphology of CA-noninteracting IN mutant viruses and the structures of HIV-1 IN bound with CA assemblies and CA hexamers. In Aim 1, we will use CA hexamers and core-like structures formed with purified CA as substrates in binding assays to determine the binding specificity and map the key domains and particular amino acids on IN involved in binding. To further confirm the IN-CA assembly interaction and to determine the oligomeric states preferred for binding and the stoichiometry and binding constants, we will use size exclusion chromatography-multi angle light scattering detector (SEC-MALS) and surface plasmon resonance (SPR) to measure IN-CA binding. We will also investigate the role of the IN-CA interaction at the core assembly step during the late phase of HIV-1 life cycle. In Aim 2, we will obtain three dimensional structures of the CA-noninteracting IN mutant viruses by cryo-electron tomography (cryo-ET), and use cryo-electron microscopy (cryo-EM) to analyze CA assemblies or CA hexamers bound with IN as another approach to examine the IN-CA interaction. The cryo-EM study may also reveal the IN binding surface on the CA multimers, which will corroborate the results obtained from biochemical and other biophysical analyses. In the process, we will shed light on the interaction between IN and CA, and the effect of such interaction on uncoating. Characterization of the interactions and determination of their biological significance may reveal new functional roles for IN, and identify new potential targets for anti-HIV therapy.